Contact Force-Guided versus Contact Force-Blinded Cavo-Tricuspid Isthmus Ablation for Atrial Flutter: A Systematic Review and Meta-Analysis

Contact force (CF) is a novel approach developed to increase the safety and efficacy of catheter ablation. However, the value of CF-sensing technology for atrial flutter (AFL) cavo-tricuspid isthmus ablation (CTIA) is inconclusive. To generate a comprehensive assessment of optimal extant data on CF for AFL, we synthesized randomized controlled trials (RCTs) and observational studies from Web of Science, SCOPUS, EMBASE, PubMed, and Cochrane until 29 November 2022, using the odds ratio (OR) for dichotomous outcomes and mean difference (MD) for continuous outcomes with a corresponding 95% confidence interval (CI). Two RCTs and three observational studies with a total of 376 patients were included in our analysis. CF-guided ablation was associated with (A) a higher rate of AFL recurrence (OR: 2.26 with 95% CI [1.05, 4.87]) and total CF (MD: 2.71 with 95% CI [1.28, 4.13]); (B) no effect on total procedure duration (MD: −2.88 with 95% CI [−7.48, 1.72]), fluoroscopy duration (MD: −0.96 with 95% CI [−2.24, 0.31]), and bidirectional isthmus block (BDIB) (OR: 1.50 with 95% CI [0.72, 3.11]); and (C) decreased radiofrequency (RF) duration (MD: −1.40 with 95% CI [−2.39, −0.41]). We conclude that although CF-guided CTIA was associated with increased AFL recurrence and total CF and reduced RF duration, it did not affect total procedure duration, fluoroscopy duration, or BDIB. Thus, CF-guided CTIA may not be the optimal intervention for AFL. These findings indicate the need for (A) providers to balance the benefits and risks of CF when utilizing precision medicine to develop treatment plans for individuals with AFL and (B) clinical trials investigating CF-guided catheter ablation for AFL to provide definitive evidence of optimal CF-sensing technology.


Introduction
Atrial flutter (AFL) is classified into typical or atypical AFL based on the cavo-tricuspid isthmus (CTI) involvement. Although typical AFL is characterized by a macro-reentrant

Eligibility Criteria
We included RCTs and observational comparative studies with the following population intervention control outcome (PICO) criteria: population (P) as patients with AFL undergoing CTIA; intervention (I) as CF-guided ablation; control (C) as CF-blinded ablation; outcome (O) as recurrence rate of AFL. The secondary outcomes include procedural outcomes (total CF, total procedure duration, fluoroscopy duration, bidirectional isthmus block (BDIB), RF duration, and the number of lesion ablations).
The exclusion criteria involved animal studies, case reports, case series, non-randomized trials, laboratory studies, and conference abstracts.

Study Selection
Two reviewers (A.R.S. and O.A.) independently screened the titles and abstracts of the articles identified in the search and assessed the full-text articles for eligibility based on predefined inclusion and exclusion criteria. Any disagreement was resolved via discussion or by a third reviewer (B.A.). The included studies were reported in a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram [8] (Appendix A).

Data Extraction
Two independent investigators (A.R.S. and O.A.) extracted the summary, baseline, and outcome data from the included studies. They extracted study characteristics (country, study design, total participants, main inclusion criteria, the primary outcome, method of AFL recurrence detection, and follow-up duration); baseline characteristics (age, gender, number of patients in each group, {congestive heart failure, hypertension, age > 75, diabetes mellitus, and prior stroke or transient ischemic attack (CHA2DS-VASc) score [10]}, left ventricular ejection fraction (LVEF), and comorbidities {history of AF, hypertension (HTN), heart failure (HF), ischemic heart disease (IHD), diabetes mellitus (DM), stroke/transient ischemic attack (TIA)}; and outcomes data (AFL recurrence, total CF, total procedure duration, fluoroscopy duration, BDIB, RF duration, and number of lesion ablations). Any disagreement was resolved via discussion or by a third reviewer (B.A.).

Risk of Bias and Quality Assessment
Two investigators, A.R.S. and O.A., independently assessed the risk of bias in the included studies using the Cochrane Collaboration's updated RoB 2 tool [11]. They evaluated six criteria: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting. Additionally, A.R.S. and O.A. employed the Risk Of Bias In Non-randomized Studies-of Interventions (ROBINS-I) tool [12] to evaluate the quality of the observational studies included. Any disagreements were resolved via discussion or with the involvement of a third reviewer, B.A.

Statistical Analysis
The Revman software version 5.4 [13] was utilized for this meta-analysis to combine dichotomous outcomes using odds ratio (OR) and continuous outcomes using mean difference (MD), accompanied by their respective 95% confidence intervals (CI). The fixed-effects model was employed for the pooled analysis, but if substantial heterogeneity was detected, the random-effects model was used instead. Heterogeneity was assessed using the chisquare test and quantified via the I-square test. Significance for the chi-square test was set at an alpha level below 0.1, and heterogeneity was considered significant if the I-square value exceeded 50%. On significant heterogeneity, sensitivity analysis by excluding one study at a time and rerunning the analysis was conducted to investigate the source of heterogeneity. Furthermore, we conducted a subgroup analysis based on the study design. Finally, we did not investigate the publication bias by funnel plots as we included less than ten studies [14].

Search Results and Study Selection
Our initial database search identified 419 records. Using COVIDence systemic review software] [15], we removed 170 duplicates and then eliminated 234 records by title and abstract screening. We then read the full text of the remaining 15 studies to finally include five studies ( Figure 1).

Statistical Analysis
The Revman software version 5.4 [13] was utilized for this meta-analysis dichotomous outcomes using odds ratio (OR) and continuous outcomes usin ference (MD), accompanied by their respective 95% confidence intervals (CI effects model was employed for the pooled analysis, but if substantial hetero detected, the random-effects model was used instead. Heterogeneity was ass the chi-square test and quantified via the I-square test. Significance for the ch was set at an alpha level below 0.1, and heterogeneity was considered signifi square value exceeded 50%. On significant heterogeneity, sensitivity analysi ing one study at a time and rerunning the analysis was conducted to investigat of heterogeneity. Furthermore, we conducted a subgroup analysis based on th sign. Finally, we did not investigate the publication bias by funnel plots as w less than ten studies [14].

Search Results and Study Selection
Our initial database search identified 419 records. Using COVIDence syst software] [15], we removed 170 duplicates and then eliminated 234 records abstract screening. We then read the full text of the remaining 15 studies to fin five studies ( Figure 1).

Characteristics of Included Studies
We included five studies [1,[15][16][17][18]: two RCTs, two prospective observational studies, and one retrospective observational study. Detailed summary characteristics of the included studies are outlined in Table 2. They were conducted in the United Kingdom, Canada, Denmark, and Australia. A total of 376 patients were included, of which 185 patients were allocated to the CF-guided group and 192 patients to the CF-blinded group. Most patients were men, including 144 (75%) men in the CF-guided group and 155 (83.7%) men in the CF-blinded. Detailed baseline characteristics of the included participants are outlined in Table 3.
It is noteworthy that the mean CF and rates of AFL recurrence varied among the included three distinct studies. In the study conducted by Begg et al. [15], we found that the total CF used in the CF-guided group was 11.4 g, accompanied by a standard deviation (SD) of 4.4. Interestingly, no cases of AFL recurrence were observed in this group, with a 0% recurrence rate at both the 3-month and 6-month checkups, mirroring results in the CF-blinded group. A comparable result was seen in Venier et al. [18], the mean CF was recorded at 13.1 g, along with an SD of 3.3. Similar to the Begg et al. study [15], the rate of AFL recurrence remained at 0%, with no cases found out of 35 subjects at both the 3-month and 6-month timepoints. Finally, Giehm-Reese et al. [1] demonstrated a mean CF of 16.7 g, with an SD of 7.5. AFL recurrence rates in this study were slightly higher, with 7 out of 66 individuals (approximately 10.6%) showing recurrence at the 3-month mark and 10 out of 58 individuals (approximately 17.2%) at the 12-month mark.

Risk of Bias and Quality of Evidence
Begg et al. [15] associated a high overall risk of bias (RoB) with a high risk of outcome measurement bias as the authors provided no information about outcome assessor blinding, randomization, and deviation from intended interventions, while Giehm-Reese et al. [1] noted significant differences in the baseline data of the participants between both groups ( Figure 2A). Also, Boles et al. [16] and Venier et al. [18] observed a moderate overall RoB, while Gould et al. [17] noted a serious overall RoB ( Figure 2B).

Primary Outcome (AFL Recurrence)
There was no difference between CF-guided and CF-blinded groups up to three months of follow up (OR: 1.52 with 95% CI [0.49, 4.74], p = 0.47); however, CF-guided ablation was associated with less AFL recurrence from 3 to 12 months of follow up (OR: 3.12 with 95% CI [1.08, 9.02], p = 0.04) ( Figure 3). Pooled studies were homogenous up to three months of follow up (I 2 = 8%, p = 0.3). However, pooled studies were heterogenous from 3 to 12 months of follow up (I 2 = 63%, p = 0.1). Hence, we used the random-effect model yielding no difference between both groups (OR: 2.69 with 95% CI [0.40, 17.99], p = 0.31). The test of subgroup difference based on the study design was not significant (p = 0.30) for up to three months (Appendix B).

Secondary Outcomes
CF-guided ablation was associated with a higher total CF (MD: 2.71 with 95% CI [1.28, 4.13], p = 0.0002) ( Figure 4A); no effect on total procedure duration (MD:    Begg et al. [15] associated a high overall risk of bias (RoB) with a high risk of outcome measurement bias as the authors provided no information about outcome assessor blinding, randomization, and deviation from intended interventions, while Giehm-Reese et al. [1] noted significant differences in the baseline data of the participants between both groups ( Figure 2A). Also, Boles et al. [16] and Venier et al. [18] observed a moderate overall RoB, while Gould et al. [17] noted a serious overall RoB ( Figure 2B).

Discussion
Based on the results of two RCTs, two prospective studies, and one retrospective study with a total of 376 patients, we conclude that CF-guided ablation is associated with (A) a higher incidence of AFL recurrence and total CF with CF-guided ablation, (B) no effect on the total procedure duration, fluoroscopy duration, or BDIB, and (C) shorter RF duration and fewer ablations per lesion. Thus, we identify characteristics of CF ablation that must be weighed by providers considering the risks and benefits of available interventions.
Our systematic review and meta-analysis until 29 November 2022, to compare CFguided ablation versus CF-blinded ablation for AFL, utilized more selective criteria to detect key features of the literature that are not identified in another systemic review and meta-analysis until June 2022 [19]. Furthermore, in our study, we included contact force alone as the primary parameter of interest. Previous analysis by Pang et al. [19] included three different contact parameters: CF, electrical coupling index (ECI), and ablation index (AI). The findings indicated that the impact of all three parameters was comparable and did not significantly contribute to the inter-group differences, except for that on fluoroscopy time [19]. In their included studies, a study involving AI showed a significant reduction in fluoroscopy time among the intervention group [20]. However, the results from the other subgroups and the overall analysis did not show a statistically significant difference [19]. Our systematic review and meta-analysis provide a more subtle quality assessment by utilizing the state-of-the-art tools (RoB 2 [11] and ROBINS-1 [12]) instead of the older tools (QUADAS-2 [21] and the Newcastle-Ottawa Scale (NOS) [22] utilized by the other systematic review and meta-analysis [19].
CTI ablation is one of the most performed ablation procedures with a low recurrence rate [2]. AFL ablation is already a highly effective and safe procedure, but several technological and methodological developments have proposed incremental improvements in efficiency without compromising safety and effectiveness. It has been proposed that a CF-guided ablation of AFL is a novel technique with the potential to reduce total RF delivery time, the time to achieve BDIB, as well as recurrence of AFL after CF ablation [15]. However, in the absence of established guidelines, additional efforts are needed to combine available data and perform pooled analysis of available studies to reach clinically applicable conclusions.
Despite the achievable endpoint of BDIB, a significant proportion of patients experience conduction recurrence via CTI, which can lead to recurrent arrhythmias. The ability to achieve a durable conduction block depends on the ability to obtain a stable, contiguous set of transmural lesions. This depends on various factors such as tissue depth, ablation electrode size, the temperature at the electrode-tissue interface, RF duration, and electrode tip tissue CF [3]. Recently, the development of CF-sensing catheters has contributed to a better understanding of the electrode tip-tissue CF relationship and subsequent lesion formation. It also enhances the contact between the electrode and tissue during RF catheter ablation, which can significantly improve procedure parameters, leading to considerable reductions in procedure duration and fluoroscopy exposure, without elevating the risk of immediate complications [23]. Considerable reductions in the occurrence of acute pulmonary vein (PV) reconnection and resting conduction have been noted during AF ablation procedures utilizing live CF data for PV Isolation. Furthermore, incorporating CF sensors for PV isolation not only decreases procedure duration but also minimizes the requirement for supplementary ablation, leading to improved long-term outcomes [24,25].
While there is growing evidence to support the significant impact of CF in assessing the effectiveness of lesions and enhancing the success rate of AF ablation, the exact role of CF guidance in CTI ablation of AFL remains uncertain. CTI is highly heterogeneous, with tissue thickness decreasing from the annulus to the vena cava, as well as the presence of multiple prominences, including ridges, pouches, or pectinate muscles. All these factors can affect the ability to create adequate ablation lesions and thus can benefit most from CF detection techniques. The results of our study indicate that CF-guided CTIA led to a significant reduction in lesion ablations but a higher recurrence rate of AFL. Based on a subgroup analysis, the recurrence rate of AFL in the first three months was not significantly different between CF-guided and CF-blinded groups. In contrast, the overall rate of AFL recurrence, as well as the rate of AFL recurrence from 3 to 12 months was significantly higher in the CF-guided group than in the CF-blinded group. This was mainly weighted by Giehm-Reese et al. [1], whose participants were older and had more comorbidities, including IHD requiring percutaneous cardiac intervention or coronary artery bypass graft as well as HTN [26]. CF-guided patients may experience a higher recurrence rate due to this overestimation.
Moreover, using contact sensing technology may assist in delivering effective ablation lesions. The ideal ablation lesion would cover the entire thickness of the myocardium with minimal collateral damage to surrounding tissue and without the generation of a "steam pop," the audible sound produced by an intramyocardial explosion when tissue temperature reaches 100 • C, resulting in gas formation [27]. Due to its association with cardiac perforation and ventricular septal defect, it is a potentially severe complication of radiofrequency ablation [28]. Therefore, the goal is to provide sufficient CF between the catheter and tissue to provide sufficient RF energy to prevent AFL recurrence but also not to cause perforation or steam pops. Frances et al. [29] and Venier et al. [18] suggested that there is an inverse correlation between RF duration and the percentage of lesions requiring greater than 10 g of CF per procedure. With an average CF of less than 10 g per procedure, the RF delivery time was significantly reduced. Furthermore, several studies have demonstrated that lesions with an average CF of <10 g have a higher risk of re-conduction after AF ablation [29]. Accordingly, 10 g might be the minimum target CF required along the CTIA to reduce the RF, fluoroscopy, and total procedure duration. CF-guided ablation was associated with a higher total CF (MD: 2.71 with 95% CI 175 [1.28, 4.13], p = 0.0002) ( Figure 4A). In both the CF-guided and CF-blinded interventions, CF-guided catheters were used. In both groups, contact force was recorded, but it was blinded to the operators in the CF-blinded group. Our study showed that the CF-guided group had a higher total CF. Excessive CF may result in complications like cardiac perforation. Achieving bidirectional block with lesser CF may be preferred to avoid such complications. Our study highlights and supports this finding since the CF-guided group achieved adequate contact force with a significant reduction in RF duration; however, we also found no difference in fluoroscopy and total procedure duration.
Radiation exposure during conventional transcatheter ablation procedures can have significant health effects, both deterministic and stochastic. Deterministic effects, such as radiation-induced skin burns, acute radiation syndrome, cataracts, sterility, and tumor necrosis, occur when a specific level of ionizing radiation exposure is reached. Stochastic effects, on the other hand, are random and probabilistic, with an extremely rare occurrence being the development of cancer in irradiated organs or tissues [30]. These effects emphasize the importance of minimizing radiation exposure whenever possible. Our study focuses on the importance of CF catheters in addressing these concerns. CF catheters facilitate improved contact and the creation of adequate ablation lesions, resulting in reduced procedure times. This reduction in procedure time is attributed to the effective and stable contact between the catheter tip and the tissue, which is crucial for both mapping and lesion formation during cardiac ablation procedures [31].
By maintaining consistent and adequate contact, CF catheters can minimize the need for repeat ablations or adjustments, leading to reduced procedure time. Inadequate contact force can result in incomplete or ineffective lesion formation, leading to the need for additional ablations. CF catheters aid in achieving optimal contact force, allowing for efficient lesion formation in a single application. This efficiency reduces the number of ablations required, thereby saving time during the procedure [32]. Furthermore, CF catheters not only provide a therapeutic approach to arrhythmias but also serve as a tool for accurately characterizing the arrhythmic substrate [33]. By providing precise and reliable contact force information, CF catheters enable clinicians to deliver optimal therapy while minimizing unnecessary energy delivery and the overall duration of the procedure, thereby reducing the need for extensive fluoroscopy time.
In light of these considerations, efforts have been made to reduce radiation exposure in electrophysiology. A study investigated contact force-controlled zero-fluoroscopy catheter ablation for right and left-sided arrhythmias, achieving a procedural success rate of 97% with minimal complications [33]. Additionally, a study from Italy evaluated physicians' awareness of radiation effects via questionnaires. The findings demonstrated satisfactory awareness but recommended further improvement [34]. It also emphasized that the awareness of radiation risks is essential for fostering a culture of respect for radiation hazards and a commitment to minimizing exposure while maximizing protection [34].
Regarding complications, adverse events were not extensively reported in the studies analyzed. In the study by Venier et al. [18], two instances of steam pops occurred in the CF-blinded group, but they had no clinical consequences. Gould et al. [17] reported a minor complication of acute groin bleeding in one patient, which resolved with rest and pressure. In the Giehm-Reese study [1], complications were reported in six patients, including groin hematomas, resulting in a delayed discharge for some patients. However, none of the hematomas required blood transfusion or surgery, with the occurrence of audible steam pops comparable between both groups. Finally, Begg et al. [15] reported an overall complication rate was 2%, with one patient experiencing a transient ischemic attack and another patient requiring treatment for ventricular fibrillation.

Limitations
First, the pooled analysis presented in this systematic review and meta-analysis is derived from two RCTs, two prospective studies, and one retrospective study, including a total of 376 patients, which is a small number that may have adversely affected our study's power. Second, the inclusion of prospective and retrospective studies increased the number of patients included in this study but also increased the RoB, particularly selection bias, since the investigators were not blinded. In addition, two RCTs [1,15] included patients who were slightly older in the intervention group, and more of them were women with higher CHA2DS2-VASc scores than the control group. Despite being attributed to chance, these differences may have affected the outcomes between intervention groups. Third, the study conducted by Giehm-Reese et al. [1] was initially powered to measure re-conduction after three months but was further extended to measure recurrent arrhythmia after 12 months.
Fourth, although theoretically, CF guidance may reduce steam pops, we acknowledge that the lack of data is a limitation of our study. However, it is worth noting that Venier et al., 2016 [18], reported no major complications, despite two steam pops occurring in the CF-blinded group, which had no clinical consequences [18]. Similarly, Giehm-Reese et al. [1] found that the number of patients with audible steam pops was similar in both the CFblinded and guided groups. Fifth, no reports of scar size were present in the RCTs included in our study. According to Begg et al., 2019 [15], CF-guided and CF-blinded techniques produced similar ablation lesions, with 33 mm being the approximate length with no significant differences between the different techniques. The results of the four RCTs included in this review demonstrated that CF-guided ablation led to a reduction in the number of lesions required to terminate AFL. Fifth, we did not investigate other parameters, including ECI or AI, which can show different findings; however, current data imply a similarity between them and CF [19]. Future investigations can be enhanced by specific measurements of scar size. Finally, there was also a disparity between the expertise of operators in CF-guided ablation across the different studies, and this could have affected their results, as CTIA is a highly precise procedure that requires highly experienced operators to provide consistent results. Also, operators may unconsciously place more ablation lesions in CF-blinded groups to promote CF-guided ablation.

Future Research Implications
We reported that the AFL rate of recurrence was higher in the CF-guided group; however, AFL recurrence rates may fluctuate, and a short-term follow up might not accurately reflect the true rates. Thus, conducting long-term follow ups can offer a more comprehensive view of recurrence rates, providing insight into the enduring effectiveness of CF-guided procedures and identifying late recurrences. Also, standardizing ablation procedure techniques, including catheter position, energy delivery parameters, lesion creation protocol, and post-procedure management, can also help minimize variability, ensure consistency, and enable the accurate evaluation of CF guidance's impact on AFL recurrence rates. Finally, operator proficiency also significantly impacts procedure outcomes, necessitating the inclusion of experienced operators in future studies. Providing standardized training and certification programs can further enhance consistency, reduce variations, and ultimately impact AFL recurrence rates. Clinical trials are needed to investigate CF-guided catheter ablation for AFL to provide definitive evidence of optimal CF-sensing technology.

Conclusions
CF-guided CTIA is associated with (A) increased risk of AFL recurrence and total CF, (B) no effect on the fluoroscopy duration, the total procedure duration, or the BDIB, and (C) reduced RF duration and the number of lesion ablations. The clinical application of CF technology in the CTIA of AFL requires further rigorous RCTs as currently available evidence is mainly derived from two small single-center RCTs and observational studies. The potentially fatal outcomes of AFL in people with heart transplantations [33] and other procedures emphasize the need for definitive studies of CF-blinded CTIA and CF-guided CTIA. This study suggests that CF-guided CTIA may not be the optimal intervention for AFL. Providers must carefully consider the adverse and beneficial effects of interventions when developing treatment plans to apply precision medicine for individuals with AFL.    Table 1 Selection process 8 Specify the methods used to decide whether a study met the inclusion criteria of the review, including how many reviewers screened each record and each report retrieved, whether they worked independently, and if applicable, details of automation tools used in the process.

Page 3 Section 2.3
Data collection process 9 Specify the methods used to collect data from reports, including how many reviewers collected data from each report, whether they worked independently, any processes for obtaining or confirming data from study investigators, and if applicable, details of automation tools used in the process. List and define all outcomes for which data were sought.
Specify whether all results that were compatible with each outcome domain in each study were sought (e.g., for all measures, time points, analyses), and if not, the methods used to decide which results to collect.

Page 3 Section 2.4 10b
List and define all other variables for which data were sought (e.g., participant and intervention characteristics, funding sources). Describe any assumptions made about any missing or unclear information.

Page 3 Section 2.4
Study risk of bias assessment 11 Specify the methods used to assess the risk of bias in the included studies, including details of the tool(s) used, how many reviewers assessed each study and whether they worked independently, and if applicable, details of automation tools used in the process.

Effect measures 12
Specify for each outcome the effect measure(s) (e.g., risk ratio, mean difference) used in the synthesis or presentation of results.

Synthesis methods 13a
Describe the processes used to decide which studies were eligible for each synthesis (e.g., tabulating the study intervention characteristics and comparing against the planned groups for each synthesis (item #5)).  Describe any methods used to assess the risk of bias due to missing results in a synthesis (arising from reporting biases).

Page 3 Section 2.5
Certainty assessment 15 Describe any methods used to assess certainty (or confidence) in the body of evidence for an outcome.

Study selection 16a
Describe the results of the search and selection process, from the number of records identified in the search to the number of studies included in the review, ideally using a flow diagram. Cite studies that might appear to meet the inclusion criteria, but which were excluded, and explain why they were excluded. Provide a general interpretation of the results in the context of other evidence. Page 11 23b Discuss any limitations of the evidence included in the review. Page 12 23c Discuss any limitations of the review processes used. Page 12 23d Discuss the implications of the results for practice, policy, and future research. Page 12

Other Information
Registration and protocol 24a Provide registration information for the review, including the register name and registration number, or state that the review was not registered.

Not available 24b
Indicate where the review protocol can be accessed, or state that a protocol was not prepared. Not available 24c Describe and explain any amendments to the information provided at registration or in the protocol.
Not available

Support 25
Describe sources of financial or non-financial support for the review, and the role of the funders or sponsors in the review.

Page 12
Competing interests 26 Declare any competing interests of review authors. Page 12 Availability of data, code, and other materials 27 Report which of the following are publicly available and where they can be found: template data collection forms; data extracted from included studies; data used for all analyses; analytic code; any other materials used in the review. and other materials studies; data used for all analyses; analytic code; any other materials used in the review. Figure A1. Forest plot of AFL recurrence for up to 3 months subgrouped by the study design [1,15,18].